European Journal of Medicinal Chemistry 222 (2021) 113588

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Research progress of dual inhibitors targeting crosstalk between

histone epigenetic modulators for cancer therapy
Ying-Chao Duan a, *, Shao-Jie Zhang a, Xiao-Jing Shi b, Lin-Feng Jin a, Tong Yu a, Yu Song a,
Yuan-Yuan Guan a, **
a
School of Pharmacy, Xinxiang Medical University, 453003, Xinxiang, Henan Province, PR China
b
Laboratory Animal Center, Academy of Medical Science, Zhengzhou University, 450052, Zhengzhou, Henan Province, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Abnormal epigenetics is a critical hallmark of human cancers. Anticancer drug discovery directed at
Received 27 March 2021 histone epigenetic modulators has gained impressive advances with six drugs available for cancer
Received in revised form therapy and numerous other candidates undergoing clinical trials. However, limited therapeutic profile,
9 May 2021
drug resistance, narrow safety margin, and dose-limiting toxicities pose intractable challenges for their
Accepted 25 May 2021
Available online 1 June 2021
clinical utility. Because histone epigenetic modulators undergo intricate crosstalk and act cooperatively
to shape an aberrant epigenetic profile, co-targeting histone epigenetic modulators with a different
mechanism of action has rapidly emerged as an attractive strategy to overcome the limitations faced by
Keywords:
Epigenetic modulator
the single-target epigenetic inhibitors. In this review, we summarize in detail the crosstalk of histone
Dual inhibitor epigenetic modulators in regulating gene transcription and the progress of dual epigenetic inhibitors
Anticancer therapy targeting this crosstalk.
Crosstalk © 2021 Elsevier Masson SAS. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Dual inhibitors targeting histone erasers and readers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1. Dual inhibitors targeting HDAC and BRD4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1.1. Crosstalk between HDAC and BRD4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1.2. Reported HDAC/BRD4 dual inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2. Dual inhibitors targeting HDAC and BRPF1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3. Dual inhibitors targeting histone erasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.1. Dual inhibitors targeting LSD1 and HDAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.1.1. Crosstalk between LSD1 and HDAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.1.2. Reported LSD1/HDAC dual inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.2. Dual inhibitors targeting LSD1 and JmjC KDM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.2.1. Crosstalk between LSD1 and JmjC KDM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.2.2. Reported LSD1/JmjC KDM dual inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4. Dual inhibitors targeting histone erasers and writers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.1. Dual inhibitors targeting HDAC and G9a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.1.1. Crosstalk between HDAC and G9a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.1.2. Reported HDAC/G9a dual inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4.2. Dual inhibitors targeting HDAC and EZH2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4.2.1. Crosstalk between HDAC and EZH2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

* Corresponding author.
** Corresponding author.
E-mail addresses: duanyingchao1986@163.com (Y.-C. Duan), 131011@xxmu.edu.
cn (Y.-Y. Guan).

https://doi.org/10.1016/j.ejmech.2021.113588
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al. European Journal of Medicinal Chemistry 222 (2021) 113588

4.2.2. Reported HDAC/EZH2 dual inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

4.3. Dual inhibitors targeting LSD1 and G9a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5. Dual inhibitor targeting histone writer and reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
5.1. Crosstalk between BRD4 and CBP/p300 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
5.2. Reported BRD4-CBP/p300 dual inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
6. Discussion and conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Declaration of competing interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

1. Introduction in other words, specific residues may undergo multiple epigenetic

modifications, leading to distinct gene expression patterns. For
Epigenetic modifications of lysine-rich N-terminal histone example, lysine residues can be targeted for various modifications,
“tails” are primarily via acetylation and methylation, ubiquitina- such as acetylation, methylation, ubiquitination, or sumoylation
tion, phosphorylation, and sumoylation. These chemical modifica- and can harbor one, two, or three methylation marks. Moreover, a
tions can associate at distinct amino acid residues with significantly specific pattern of histone modifications frequently affects the
divergent transcriptional information. For example, H3K9 acetyla- generation or loss of other modifications, demonstrating extensive
tion and H3K4, H3K36, and H3K79 methylation are associated with connections, and crosstalk between histone modifications. For
open chromatin and gene transcription, whereas methylation of instance, acetylation on H3K4 or H3K9 and phosphorylated H3S10
H3K9, H3K27, and H4K20 suppress gene expression [1]. These or H3T11 can block the G9a activity, leading to decreased H3K9
epigenetic modifications of histone are reversible and highly gov- methylation levels. The increased acetylation level for residues
erned by three categories of epigenetic modulators: “writers”, proximal to H3K4 contributes to repressing LSD1 activity and
“erasers”, and “readers”. “Writers” mediate epigenetic modifica- subsequent H3K4 methylation enhancement, whereas sumoylated
tions transfer to the histone tails, while “erasers” remove such H4 stimulates intranucleosomal demethylation by LSD1 [11].
chemical modifications, and “readers” recognize and bind to these Given that various histone epigenetic modulators undergo
specific epigenetic marks. Histone acetyltransferases (e.g., CBP/ intimate interplay and act in a combined manner to activate or
p300) and histone methyltransferases (e.g., EZH2, G9a, and DOT1L) repress chromatin-mediated transcription, dual inhibitors target-
represent well-known “writers”. Histone deacetylases (e.g., HDAC, ing different epigenetic modulators is an effective strategy to
SIRT) and lysine demethylases (e.g., LSD1, JmjC KDM) represent improve single epigenetic-target agents' safety and efficacy and
“erasers”; “readers” largely fall into three families: bromodomain- overcome resistance [12,13]. In this review, we discussed the
containing proteins, methyl-lysine, and methyl-arginine-binding crosstalk of histone epigenetic modulators in regulating gene
domain-containing proteins, and PDH-containing proteins [2,3]. transcription, tumorigenesis and the recent development of dual
Epigenetic regulation plays a crucial role in many cellular pro- inhibitors targeting this crosstalk, compounds targeting multiple
cesses, including proliferation, differentiation, cell cycle, apoptosis, isoforms from the same family will not be discussed in this article.
and others. Dysfunction of epigenetic modulators is a common
feature of cancer development and progression, making these 2. Dual inhibitors targeting histone erasers and readers
epigenetic modulators attractive targets for cancer therapy [4e6].
Alternative to the irreversible genomic mutations that inactivate 2.1. Dual inhibitors targeting HDAC and BRD4
tumor suppressor genes or activate oncogenes in cancer, the
reversible nature of these epigenetic modifications confers a 2.1.1. Crosstalk between HDAC and BRD4
unique therapeutic advantage to targetingepigenetic modulators Histone deacetylases (HDAC) are a family of epigenetic enzymes
for cancer treatment. Aberrant histone modifications, including responsible that remove acetyl marks of lysine residues in histones
(de) acetylation and (de) methylation of lysine residues, are the and some non-histone proteins, including tumor suppressor p53,
most characterized and pharmaceutically used epigenetic changes. microtubules, and Hsp90 [14,15]. Histone lysine deacetylation re-
To date, six drugs targeting histone epigenetic modulators have stores lysine's positive charge and enhances histone's affinity for
been approved: five for HDAC inhibitors and one for EZH2 in- DNA, leading to a more condensed form of chromatin and gene
hibitors [7]. Furthermore, numerous small inhibitors are directed at silencing [16]. HDAC plays a crucial role in regulating multiple
distinct histone epigenetic modulators in clinical trials against processes of life, from cell cycle, cell proliferation, cell differentia-
various forms of cancer. A list of histone epigenetic modulators tion, and apoptosis to protein activities. Numerous correlative
under investigation is presented in Table 1. Despite the impressive studies have demonstrated aberrant HDAC expression in human
advances in epigenetic drug discovery, there are challenges and cancers, making HDAC an intently pursued target [17]. Starting with
limitations to overcome, including the following: (1) most Vorinostat (SAHA), the first HDAC inhibitor approved by the FDA
currently approved epigenetic inhibitors are restricted to treating (U.S. Food and Drug Administration) in 2006 for treating refractory
hematological malignancies, their efficacy in solid tumors remains cutaneous T-cell lymphoma, developing HDAC inhibitors for cancer
uncertain; (2) epigenetic agents often have a narrow safety margin, therapies has attracted tremendous efforts and resulted in
and dose-limiting adverse events such as the thrombocytopenia impressive successes with five HDAC inhibitors approved for clin-
and gastrointestinal toxicities are observed; (3) epigenetic agents ical use, four approved by FDA: Vorinostat (SAHA), Romidepsin
frequently require a longer time to exert their clinical response (FK228), Belinostat (PXD101), Panobinostat (LBH589), and one
compared with many other targeted agents; (4) intrinsic drug approved by CFDA (China Food and Drug Administration): Tucidi-
resistance to some epigenetic inhibitors such as HDAC and BRD4 nostat (also known as Chidamide). Also, other numerous HDAC
inhibitors have been described [8e10]. inhibitors are in the clinical trial stage [18].
Histone epigenetic modulators share many common residues; The canonical pharmacophore features of HDAC inhibitors
2
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al. European Journal of Medicinal Chemistry 222 (2021) 113588

Table 1
Approved and clinical trial drugs targeting histone epigenetic modulators.

Group Modulators Category Epigenetic target Approved Drugs Drugs in clinical trials
function

Histone HDACs Eraser Remove acetyl groups Tucidinostat (Chidamide), Abexinostat, Fimepinostat, Resminostat, Mocetinostat, Pracinastat,
Acetylation from histone lysines Vorinostat, Belinostat, Entinostat, Quisinostat, Givinostat, Tefinostat, Tinostamustine,
Romidepsin, Panobinostat Citarinostat, Ricolinostat, Domatinostat (4SC-202)
Trichostatin A, Bisthianostat, CUDC-101, OBP-801, CG200745, CXD101,
KA2507, OKI-179, NBM-BMX, AR-42,
SIRT1 Eraser Remove acetyl groups e Selisistat
from histone lysines
BET Reader Read acetyl groups on e BMS-986158, CPI-0610, GSK2820151, Mivebresib, MK-8628, Molibresib,
histone lysines Alobresib, RG6146, RVX000222, ZEN003694, ABBV-744
BRPF1 Reader Read acetyl groups on e e
histone lysines
CPB/p300 Writer Acetylate histone e CCS1477
lysines
Histone LSD1 Eraser Remove methylation e GSK2879552, IMG-7289, INCB0598725, TAK-418, ORY-2001, CC-90011,
marks from H3K4 and Seclidemstat (SP-2577),
Methylation H3K9
EZH2 Writer Methylates histone Tazemetostat CPI-0209, CPI-1205, GSK2816126, DS-3201b, HH2853, SHR2554, PF-
H3K27 06821497
EED Writer Methylates histone e MAK683
H3K27
DOT1L Writer Methylates histone e Pinometostat
H3K79
G9a Writer Methylates histone e e
H3K9
PRMT Writer Methylates histone e GSK3368715, GSK332659, JNJ-64619178, PF-06939999, PRT811
arginines

consist of three parts, as shown in Fig. 1: a surface recognition cap
capable of interacting with the hydrophobic rim at the entrance of
the active pocket, a zinc-binding group (ZBG) that chelates zinc ion
by forming coordinate bonds within the active site, and a linker that
connects the cap and ZBG and interacts with the hydrophobic
tunnel of the active site. The most commonly used ZBG groups are
the hydroxamic acid and ortho-amino anilide group, while romi-
depsin uses a thiol group to coordinate the active site's zinc ion.
Other ZBGs, including 3-hydroxypyridin-2-thione [19] and dithio-
carbonate [20] are reported. Extensive structure-activity relation-
ship (SAR) studies confirm that the cap group in HDAC inhibitors is
flexible and tolerates modification. Thus, ZBG can be easily linked to
other anticancer scaffolds providing numerous potent multi-target
inhibitors [12,21,22].
The bromodomain and extra-terminal (BET) domain family are
epigenetic readers featuring a tandem repeat of bromodomains

Fig. 1. Pharmacophore features and structures of approved HDAC inhibitors.

3
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
(BD1 and BD2) at their N-terminus. The BET family includes four
protein members (BRD2, BRD3, BRD4, and BRDT), and BRD4 is the
most characterized member of this family [23,24]. BRD4 prefer-
entially binds acetylated lysine on histone's tail through their
tandem bromodomains to recruit transcription factors, including P-
TEFb, a global transcriptional elongation factor important for most
RNA polymerase II (Pol II) transcription. This (BRD4) also regulates
the transcription of a vast array of inducible genes essential for
multiple cellular processes [25,26]. BRD4 can also bind non-histone
proteins in a bromodomain-independent manner, such as the
binding of BRD4 to FLI1, MYB, SPI1, CEBPA/B, or p53 [27]. Moreover,
BRD4 also shows an intrinsic kinase activity, a function that is not
elucidated [28]. Deregulated BRD4 protein is strongly associated
with the occurrence and progression of several malignant human
diseases, especially cancer. BRD4 is implicated in the expression of
many well-known oncogenes, including c-MYC, MYCN, FOSL1, and
BCL-2, leading to considerable drug discovery efforts against this
target [29e31]. Currently, many BRD4 inhibitors have been
discovered and reported, and some of them are undergoing
different clinical trial phases for patients with solid tumors and
hematologic malignancies, such as BMS-986158, CPI-0610, Mive-
bresib (ABBV-075), Alobresib (GS-5829), ZEN003694, ABBV-744,
and others [13,32,33]. Preliminary results from clinical trials
confirm the anticancer potential of BRD4 inhibitors, but their effi-
cacy as single agents against solid tumors seems to be limited.
Frustratingly, intrinsic and acquired drug resistance and adverse
events have been observed [10,34,35].
HDAC and BRD4 share many common acetylation marks of
lysine residues on histone tails, such as H3K4 and H3K9. So, it is not
surprising that there exists an intimate interplay between HDAC
and BRD4. HDAC condenses chromatin structure by removing
acetylation “marks” on histone, which need to be recognized by the
BRD4 before facilitating the activation of specific target genes. The
global acetylation profile of the genome regulated by HDAC is
closely related to the recruitment and location of the BRD4 protein
(Fig. 2).
Direct or indirect functional interplay between HDAC and BRD4
has been reported. Fu et al. demonstrated that N-CoR/HDAC3
complex suppressed P-TEFb activity and consequently transcrip-
tional elongation through interaction with HEXIM1 to deacetylate
cyclin-dependent kinase 9 (CDK9), an internal component of P-
TEFb, thus counteracting the BRD4-mediated transcriptional acti-
vation effect [36].
Signal transducer and activator of transcription 5 (STAT5) are
essential regulators of cell differentiation, proliferation, and sur-
vival. A research work from Rascle's lab indicated that BRD2 was
required to efficiently recruit the transcriptional machinery at
STAT5 to activate its target genes [37]. Following HDAC inhibitor
treatment, delocalization of BRD2 from soluble chromatin to
insoluble chromatin and subsequent STAT5-mediated transcription
suppression was observed. Chen's group identified that upon stress
Fig. 2. Molecular crosstalk model between HDAC and BRD4. BRD4 recognizes acetylated lysine on the nucleosomal histones and recruits P-TEFb to facilitate RNA polymerase II-
dependent transcription elongation. HDAC can affect the location and functions of BRD4 by changing the global acetylation profile of nucleosomal histones.
4
European Journal of Medicinal Chemistry 222 (2021) 113588
stimuli, protein phosphatase PP1a dephosphorylated H3S10ph,
which facilitated the class I HDAC-mediated deacetylation of
nucleosomal H4K5ac/K8ac. This released chromatin-bound BRD4
for subsequent recruitment of P-TEFb to promote the target gene
expression [38]. In pancreatic ductal adenocarcinoma (PDAC) cell
lines, HDAC inhibitor 4SC-202 attenuated TGF b-induced gene
repression and epithelial-to-mesenchymal transition in a BRD4-
and c-Myc-dependent manner [39].
Recent studies revealed that the combination of HDAC and BRD4
inhibitors showed synergistic anticancer activities in different tu-
mor cell models (Table 2). Fiskus et al. found that cotreatment with
BRD4 antagonist, (þ)-JQ1, and HDAC inhibitor, Panobinostat, syn-
ergistically inhibited the growth of both cultured acute myeloid
leukemia (AML) cells and primary AML blast progenitor cells
expressing mutant NPM1cþ or MLL fusion oncoprotein, but not of
normal CD34cþ hematopoietic progenitor cells. Compared with
treatment with a single drug, combined treatment with (þ)-JQ1
and Panobinostat exerted a superior effect in prolonging the sur-
vival of non-obese diabetic/severe combined immunodeficient
(NOD/SCID) mice engrafted with OCI-AML3 or MOLM13 cells [40].
Mazur et al. observed that combined inhibition of BRD4 and HDAC
using (þ)-JQ1 and SAHA exerted a potent synergistic tumor-
suppressive effect on PDAC cells in vitro and in vivo, without evi-
dence of tumor relapse or metastasis [41]. Geng's group showed
that HDAC inhibitors facilitated BRD4-mediated transcriptional
upregulation of leukemia inhibitory factor receptor (LIFR) by
increasing histone acetylation at the LIFR promoter. This led to
feedback activation of JAK1-STAT3 signaling and attenuated the
efficacy of HDAC inhibitors in breast cancers. Conversely, blocking
the LIFR-JAK1-STAT3 signal with BRD4 inhibitor potentiates HDAC
inhibitor activity in breast cancer [42]. Zhao's group also confirmed
combining BRD4 and HDAC inhibition a potential therapy for breast
cancer. Mechanistically, combination treatment with (þ)-JQ1 and
Mocetinostat attenuated the Ras/MEK/ERK pathway through up-
regulating USP17, leading to reduced breast cancer cell viability
[43]. Zhang et al. illustrated that Panobinostat and OXT-015 com-
bination elicited a pronounced synergistic reduction in cellular
viability across a broad range of glioblastoma (GBM) model systems
and exerted antiglioma activity in GMB xenograft models [44].
Meng et al. also found that cotreatment with Panobinostat and
OXT-015 or (þ)-JQ1 caused improved repression of GBM-associated
oncogenic genes and induction of GBM-associated tumor-sup-
pressive genes compared with individual drug treatment, contrib-
uting to a highly synergistic antiproliferative and proapoptotic
effect [45]. Zhao et al. demonstrated that co-administration of
BRD4 inhibitor with HDAC inhibitor synergized to reduce the
expression of proliferative drivers such as c-Myc, cyclin D1, and
NFkB, promote apoptosis and achieved superior anticancer effects
against cutaneous T-cell lymphoma [46]. In B-cell lymphoma cells,
Romidepsin suppressed BCL6 function by promoting BCL6 acety-
lation and subsequent BCL6 downregulation, resulting in
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
Table 2
Published combination trials of HDAC and BRD4 inhibitors for cancer treatment.
HDAC inhibitor BRD4 inhibitor
Panobinostat (þ)-JQ1
SAHA (þ)-JQ1
LBH589 I-BET151
SAHA or Mocetinostat (þ)-JQ1
SAHA (þ)-JQ1
Romidepsin (þ)-JQ1
JNJ-26481585 (þ)-JQ1
Romidepsin (þ)-JQ1
Panobinostat OTX015 or (þ)-JQ1
SAHA (þ)-JQ1
SAHA (þ)-JQ1
significant cell apoptosis and cell cycle arrest. When used combined
with (þ)-JQ1, strong synergistic effects on proliferation and
apoptosis were observed [47]. The combined therapeutic efficacy of
(þ)-JQ1 with various HDAC inhibitors was also validated in rhab-
domyosarcoma (RMS) cells [48]. An evident synergistic interaction
of various HDAC inhibitors (i.e., JNJ-26481585, SAHA, LBH589, and
MS275) and (þ)-JQ1 to trigger apoptosis in RMS cells was discov-
ered. Notably, (þ)-JQ1 and JNJ-26481585 cooperatively suppressed
colony formation and triggered cell death in an in vivo RMS model,
more effective than either treatment alone. Liu et al. showed that
simultaneous inhibition of BRD4 and HDAC with (þ)-JQ1 and SAHA
synergistically suppressed gallbladder cancer cell lines' prolifera-
tion by downregulating BRD4 expression and blocking PI3K/AKT
and MAPK/ERK pathways. In in vivo efficacy evaluation of NOZ
xenograft model, cotreatment with (þ)-JQ1 and SAHA exerted a
higher efficacy than (þ)-JQ1 or SAHA used alone [49]. Hoffmann's
group identified a significant synergistic interaction between
(þ)-JQ1 and Romidepsin to induce apoptosis in urothelial carci-
noma cell lines, with reduced toxic side effects in normal cells [50].
In addition, combined inhibition of BRD4 and HDAC also syner-
gistically suppressed melanoma and c-Myc-induced murine lym-
phoma growth [51,52].
These findings suggest that the design of dual HDAC/BRD4 in-
hibitors could be a potentially effective therapy for cancers, and
medicinal chemists are dedicating considerable efforts to develop
dual HDAC/BRD4 inhibitors.
2.1.2. Reported HDAC/BRD4 dual inhibitors
As the first reported potent pan-BET inhibitor, (þ)-JQ1 has been
widely used as a tool molecule or positive control to explore BET
proteins' biological function [24]. The co-crystal structure of
(þ)-JQ1-BRD4 complex showed that the tert-butoxycarbonyl group
was oriented toward the solvent region, making it suitable for
chemical modification, which extensive SAR explorations have
validated. Sheng's group discovered a series of novel HDAC/BRD4
dual inhibitors, where hydroxamic acid moiety as the ZBG was
introduced to the pharmacophore of (þ)-JQ1 by replacing the tert-
butoxycarbonyl with a N-phenylacetamide (Fig. 3) [53]. Among
them, compound 1 exhibited potent and well-balanced inhibitory
activities against BRD4 BD1 (IC50 ¼ 11 nM) and HDAC1
(IC50 ¼ 21 nM), which was translated into its greater anticancer
potency against capan-1 cells (IC50 ¼ 0.15 mM) than (þ)-JQ1
(IC50 ¼ 8.6 mM) and SAHA (IC50 ¼ 5.4 mM) used alone or in com-
bination (IC50 ¼ 1.0 mM) by synergistic HDAC1/BRD4 dual inhibi-
tion. Compound 1 displayed pan-BET inhibitory activities with IC50
values in the nanomolar range and showed a high selectivity
against four tested non-BET BRDs, similar to that of (þ)-JQ1. HDAC
profiling revealed that compound 1 exhibited a robust inhibition
against the other four tested HDAC isoforms (HDAC2, 3, 6, and 8)
with IC50 values ranging from 21 to 192 nM, showing no obvious
5
European Journal of Medicinal Chemistry 222 (2021) 113588
Cancer type Reference
AML [40]
Murine lymphoma [52]
Melanoma [51]
Breast cancer [42,43],
Pancreatic adenocarcinoma [41]
B-cell lymphoma [47]
Rhabdomyosarcoma [48]
Urothelial carcinoma [50]
Glioblastoma [44,45],
Gallbladder cancer [49]
Cutaneous T-cell lymphoma [46]
HDACs isoform-selectivity. Consistent with the observed in vitro
effects, in human Capan-1 pancreatic cancer xenograft model,
compound 1 exerted an excellent in vivo anticancer efficacy and a
tumor growth inhibition (TGI) of 69% (bid, 15 mg/kg) and 87.7% (bid,
20 mg/kg), respectively, more potent than that of (þ)-JQ1
(TGI ¼ 41%) and SAHA (TGI ¼ 49%) used alone or in combination
(TGI ¼ 57%). This illustrates that simultaneously targeting HDAC/
BRD4 is beneficial over single-target inhibition. RT-qPCR, immune
fluorescence, and western blotting assays revealed that in tumor
tissues, c-Myc and CDC25b mRNA expression was downregulated.
In contrast, Ac-H3 and Ac-H4 expression was increased, indicating
that BRD4 and HDAC were effectively inhibited in vivo.
By rationally fusing the pharmacophore of (þ)-JQ1 with well-
established HDAC inhibitors such as CI994, SAHA, and Panobino-
stat, Fulda's group discovered several new HDAC/BRD4 dual in-
hibitors. Among them, compound 2 (termed TW09) obtained by
substituting the tert-butyl ester of (þ)-JQ1 by a class I selective
HDAC inhibitor CI994, was identified as the most active compound
with IC50 values of 729 and 74 nM against BRD4 BD1 and BD2,
respectively, displaying approximately 10-fold higher affinity for
BRD4 BD2 than BD1. The binding constants determined by
isothermal titration calorimetry showed that TW09 has a high
binding affinity to BRD4 BD1 (Kd ¼ 69 nM) similar to (þ)-JQ1
(Kd ¼ 51 nM) but is less potent than (þ)-JQ1 in binding to BDR4 BD2
(Kd ¼ 230 nM). TW09 was also a potent inhibitor of HDAC1 with an
IC50 value of 290 nM, more potent than the parental HDAC inhibitor
CI994, which had an IC50 of 960 nM under the same conditions
(Fig. 4) [54]. The co-crystal structure of TW09 with BRD4 BD1
revealed that TW09 bound to BRD4 BD1 in a very similar pattern to
(þ)-JQ1. TW09 formed key hydrogen-bond interactions between
triazole moiety with evolutionarily conserved residue Asn140,
leaving the HDAC inhibitor pharmacophore in a solvent-exposed
region, possibly accounting for its potent HDAC/BRD4 dual inhibi-
tion potency [55]. In RMS cells, TW09 could efficiently modulate
the expression of BRD4 and HDAC target proteins, including c-Myc
and Ac-H3, induce apoptosis, block colony formation, and display
similar antiproliferative activity at the same concentration
compared with (þ)-JQ1 and MS-275 used in a 1:1 combination.
Further antitumor mechanism studies showed that TW09 triggered
significant mitochondrial apoptosis by up-regulating proapoptotic
proteins (BIM, NOXA, PUMA, and BMF), downregulating anti-
apoptotic protein BCL-XL, and then activating BAK, BAX, and cas-
pase3/9, thereby exerting its potent anticancer activity. TW09 also
displayed robust antiproliferative activity against a panel of
pancreatic cancer cell lines (MIA PaCa-2, DAN-G, and HPAC), more
potent than (þ)-JQ1, CI994 used alone or incombined treatment
[55]. More significantly, compared with single-agent or combined
treatment with (þ)-JQ1 and CI994, TW09 showed more sustained
growth-inhibitory effects in a long-term proliferation assay. Inter-
estingly, TW09 shared a very similar chemical structure with
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
Fig. 3. HDAC/BRD4 dual inhibitor based on (þ)-JQ1.
Fig. 4. HDAC/BRD4 dual inhibitor based on (þ)-JQ1 and CI994.
compound 1. Structurally, the only difference between these two
molecules is replacing the hydroxamic acid group in compound 1
with an ortho-amino anilide moiety. However, TW09 demon-
strated class I HDAC isoform selectivity, while compound 1 was a
potent pan-HDAC inhibitor.
Recently, He's group designed and synthesized several selective
HDAC/BRD4 dual inhibitors based on thieno[2,3-d]pyrimidine
compound 3, a novel BRD4 inhibitory scaffold (IC50 ¼ 0.85 mM)
discovered by fragment-based design strategy (Fig. 5) [56]. The
most potent compound, 4, simultaneously inhibited BRD4 and
HDAC2/6 with IC50values of 710 nM, 58 nM, and 73 nM, respec-
tively. SAR analysis indicated that the connection position of ZBG,
not the ZBG and linker moiety, was crucial for BRD4 inhibitory
activity. For example, compound 5 with the ZBG moiety on the
piperidine ring was inactive against BRD4. HDAC isoform selectivity
assay showed that compound 4 exhibited good selectivity toward
class I and class IIb HDAC (IC50: 0.05e0.97 mM) over class IIa and IV
HDAC (IC50 > 10 mM). Compound 4 also displayed a high selective
profile toward BRD4 over other isoforms of BRD proteins. At the
cellular level, compound 4 potently suppressed the growth of three
tested colorectal carcinoma cell lines including HCT-116
6
European Journal of Medicinal Chemistry 222 (2021) 113588
(IC50 ¼ 0.45 mM), SW620 (IC50 ¼ 1.78 mM), and DLD1
(IC50 ¼ 2.11 mM), better than SAHA and RVX208 used alone or
combined. Compound 4 efficiently regulated c-Myc and Ac-H3
expression, blocked the cell cycle, promoted apoptosis, and
induced autophagy in HCT-116 cancer cells. In the HCT-116 xeno-
graft model, oral administration of compound 4 effectively inhibi-
ted HDAC/BRD4, induced autophagic cell death, suppressed IL6-
JAK-STAT signaling pathways, and showed superior in vivo anti-
tumor potencies than SAHA with TGI of 42.7% (15 mg/kg) and 68.8%
(30 mg/kg), respectively. No significant signs of toxicity were
observed in the main organs. Also, pharmacokinetics and pre-
liminary safety studies showed that compound 4 had a reasonable
pharmacokinetic profile and oral bioavailability of 40.5% in rats.
ABBV-744 is a highly potent BD2-selective BRD4 inhibitor with
Kd values of 3300 nM and 2.1 nM for BRD4 BD1 and BD2, respec-
tively, being tested in phase I trial for relapsed/refractory AML
cancer and myelofibrosis [57,58]. The co-crystal structure of ABBV-
744 with BRD2 revealed that the ethyl amide moiety is located at
the solvent-oriented region and plays an essential role in differ-
entiating BD1 and BD2 together with 2,6-dimethylphenyl ether
moiety.
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
Fig. 5. HDAC/BRD4 dual inhibitors based on thieno[2,3-d]pyrimidine BRD4 inhibitors.
Inspired by these pieces of evidence, Hu's group designed and
synthesized various novel HDAC/BRD4 dual inhibitors by merging
the common pharmacophore of HDAC inhibitors to the ethyl amide
moiety of ABBV-744 (Fig. 6) [59]. Interestingly, by introducing
different zinc-binding groups to ethyl amide moiety and exploring
substituents on the 2-position of the phenyl ring, three dual in-
hibitors with different subtype selectivity were identified. Among
them, compound 6 simultaneously inhibited HDAC1
(IC50 ¼ 59.7 nM), BRD4 BD1 (IC50 ¼ 5.6 nM), and BRD4 BD2
(IC50 ¼ 2.1 nM) but possessed no obvious activity against HDAC4/6/
8/11 (IC50 > 10 mM), thereby defined as a HDAC1/BRD4 dual in-
hibitor. Consistent with its HDAC subtype selectivity, compound 6
significantly upregulated the expression level of Ac-H3 in MV-4-
11 cells, rather than Ac-tubulin, a physiological substrate of HDAC6.
Also, compound 6 could potently attenuate c-Myc expression in
MV-4-11 cells, leading to G0/G1 cell cycle arrest, apoptosis induc-
tion, and stronger inhibitory activity (IC50 ¼ 40 nM) than MS-275
(IC50 ¼ 260 nM), ABBV-744 (IC50 ¼ 300 nM), and OTX-015
(IC50 ¼ 150 nM). Compound 7 was identified as a pan-HDAC/
BRD4 dual inhibitor, exhibiting nanomolar potency against
HDAC1/3/6/8, BRD4 BD1 (IC50 ¼ 29.5 nM), and BRD4 BD2
(IC50 ¼ 6.0 nM). In MV-4-11 cells, compound 7 showed comparable
antiproliferative activity, apoptosis induction, and cell cycle-
blocking effect compared with compound 6. Compound 8 showed
a good potency for HDAC6 (IC50 ¼ 17.2 nM) and BRD4 BD2
(IC50 ¼ 1.2 nM), with a 13- and 300-fold selectivity over HDAC1
(IC50 ¼ 228.3 nM) and BRD4 BD1 (IC50 ¼ 368.7 nM), respectively,
making it a HDAC6/BRD4 BD2 dual inhibitor. Compound 8
demonstrated potent inhibitory activity against MV-4-11 cells with
an IC50 value of 0.3 mM; however, unlike compounds 6 and 7, it
could not effectively induce G0/G1 phase arrest at a concentration
of 1.0 mM, similar to the BD2-selective BET inhibitor ABBV-744.
Several tetrahydroquinoline (THQ) derivatives, including I-
7
European Journal of Medicinal Chemistry 222 (2021) 113588
BET295, have been described as BET inhibitors. In this compound,
the THQ acetamide motif exactly mimics the acetyl-lysine substrate
in binding with BRD4. The 4-isopropylcarbamate substituent forms
a hydrophobic interaction with the WPF shelf region, and the para-
position of the 6-arylsubstituent, which protrudes into the ZA
channel, is solvent-exposed. Thus, compound 9 was designed as a
dual HDAC/BRD4 inhibitor by incorporating the polymethylene
hydroxamate chain typical of SAHA to the para-position of the 6-
arylsubstituent (Fig. 7). Compound 9 showed potent inhibition
against BRD4 (IC50 ¼ 50 nM) and HDAC1/6 (IC50 ¼ 250 nM and
420 nM). In cells, compound 9 inhibited cell proliferation in NUT
midline carcinoma (NMC) and AML cells and reduced TNF-a and IL-
6 production from LPS-stimulated peripheral blood mononuclear
cells.
Dimethylisoxazole is another well-known effective scaffold of
BRD4 inhibitor [60]. As a good starting point, several novel 3,5-
dimethylisoxazole derivatives with a hydroxamate group were
synthesized and identified as HDAC/BRD4 dual inhibitors by Chen's
group [61]. Considering the similarity between the ZA channels of
BRD4 and tubular channel of HDAC, the hydroxamic acid group was
connected to the 1-position of BRD4 inhibitor compound 10
through a flexible carbon chain, hoping to increase the ability to
occupy the ZA channel while achieving HDAC inhibitory activity
(Fig. 8). Two representative compounds, 11 and 12 showed robust
inhibitory activity against HDAC1 (IC50 ¼ 0.27 and 0.15 mM,
respectively) and BRD4 (IC50 ¼ 0.85 and 0.67 mM, respectively).
Temperature shift experiments showed that compounds 11 and 12
had a good affinity with BRD4 and exhibited a certain selectivity for
the other five tested bromodomain proteins. In vitro anticancer
assays revealed that compounds 11 and 12 strongly repressed the
growth of K562 and MV4-11 cancer cell lines, with IC50 values of
0.91e2.33 mM, slightly weaker than the positive control drugs,
(þ)-JQ1 and SAHA.
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al. European Journal of Medicinal Chemistry 222 (2021) 113588

Fig. 6. HDAC/BRD4 dual inhibitors based on ABBV-744.

Fig. 7. HDAC/BRD4 dual inhibitors based on I-BET295.

Recently, Liu's group also reported a series of novel was the most active against BRD4 with 88% inhibition at 10 mM.
dimethylisoxazole-based HDAC/BRD4 dual inhibitors by merging Compound 14 also showed potent inhibition against HDAC1,
the core structural scaffold of a reported BRD4 inhibitor, P-0014, HDAC2, and HDAC3, rather than HDAC6 (IC50 > 1.0 mM), with IC50
and an HDAC inhibitor compound 13 (Fig. 8) [62]. Compound 14 values of 0.18 mM, 0.30 mM, and 0.005 mM, respectively. In THP-

8
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
Fig. 8. HDAC/BRD4 dual inhibitors based on dimethylisoxazole BRD4 inhibitors.
1 cells, compound 14 dose-dependently downregulated c-Myc
expression and promoted Ac-H3 expression, indicating that com-
pound 14 was an effective HDAC/BRD4 dual inhibitor at the cellular
level.
Based on the reported N6-benzoyladenine BRD4 inhibitors,
Seika et al. designed and synthesized various novel HDAC/BRD4
dual-target inhibitors (Fig. 9) [63]. Compound 15 showed the
strongest HDAC/BRD4 dual-inhibitory activity, with IC50 values of
0.26 and 2.7 mM. In HL-60 cancer cells, compound 15 can inhibit cell
proliferation (GC50 ¼ 4.4 mM), time-dependently induce apoptosis,
and enhance ATRA-induced cell differentiation (EC50 ¼ 9.4 mM).
Importantly, compound 15 also exhibited growth-inhibitory activ-
ity against several BRD4 inhibitor-resistant cancer cells, including
K-562 (GC50 ¼ 7.1 mM), T-47D (GC50 ¼ 2.3 mM), and OVCAR-5
(GC50 ¼ 18 mM) cell lines.
Shao et al. discovered various novel HDAC/BRD4 dual inhibitors,
where the hydroxamic acid moiety as the zinc-binding functional
group was introduced to the free hydroxyl group of the BRD4 in-
hibitor, RVX-OH, through various linkers (Fig. 9). The most prom-
ising compound, 16, had an IC50 value of 0.20 mM against HDAC1.
Compound 16 also showed potent activity toward BRD4 BD2
(IC50 ¼ 0.40 mM), but not BRD4 BD1 (IC50 > 5.0 mM). Compound 16
showed efficient antiproliferative activities against three AML cell
lines (MV4-11: IC50 ¼ 0.56 mM, OCI-AML2: IC50 ¼ 0.38 mM, and OCI-
AML3: IC50 ¼ 0.43 mM) with greater potency than the reference
drugs, RVX-208 and SAHA. Western blotting revealed that
9
European Journal of Medicinal Chemistry 222 (2021) 113588
compound 16 showed a more dramatic down-regulation effect on
c-Myc level than SAHA and (þ)-JQ1 in OCI-AML2 and OCI-AML3
cells at 1.0 mM [64].
2.2. Dual inhibitors targeting HDAC and BRPF1
BRPF1 (the bromodomain- and PHD finger-containing protein 1)
belongs to the subfamily IV of the human bromodomains and is a
unique multivalent epigenetic “reader” harboring three histone-
binding domains: one bromodomain for recognizing acetyl-lysine
marks, two PHD fingers for binding to unmodified histone H3,
and a PWWP domain for specific interaction with methylated his-
tone H3. BRPF1 associates with three histone acetyltransferases,
including MOZ (monocytic leukemic zinc finger), MORF (MOZ-
related factor), and HBO1 (HAT bound to ORC1), assembling a
tetrameric acetyltransferase complexe that acetylate histones and
regulate transcriptional programs. In the tetrameric complex,
BRPF1 serves as a central scaffolding protein to link subunits
together into the functional complex, restrict substrate specificity,
and enhance acetyltransferase activity [65]. Various mutations in
BRPF1 have been implicated in acute leukemias, and therapeutic
targeting of BRPF1 by small molecules is emerging as an attractive
strategy for AML therapy [66].
Compounds 17e18 were synthesized and identified as potent
BRPF1 inhibitors starting with the fragment hit N-methylquinolin-
2(1H)-one, which had an IC50 of 4.8 mM for BRPF1 [67]. Compounds
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
Fig. 9. Other reported HDAC/BRD4 dual inhibitors.
17e18 potently inhibited BRPF1 with IC50 values of 7.9 and 60 nM,
respectively. Co-crystal structure of 18 with BRPF1B displayed that
compound 18 occupied the conserved acetyl methyl pocket of
BRPF1 and the 1,4-dimethylquinolin-2-one scaffold served as the
acetyl-lysine mimetic with the carbonyl forming a hydrogen bond
with the conserved Asn708 and forming an indirect hydrogen bond
with Tyr665 mediated by a water molecule. Structure guided
optimization of benzhydroxamic acids by Heimburg et al. led to a
series of highly potent and selective HDAC8 inhibitors [68]. Com-
pounds 19 was one of the most potent and selective HDAC8 in-
hibitor with an IC50 value of 69 nM. Detailed SAR exploration
showed that the two-atom linkers between benzhydroxamic acid
moiety and aromatic cap group and the meta-substitution pattern
were crucial for the HDAC8 selectivity.
Based on the available co-crystal structure of BRPF1 and com-
pound 18, and the reported selective HDAC8 inhibitors 19, com-
pounds 20e21 were designed and synthesized using a
pharmacophore fusion approach (Fig. 10). The biochemical and
biophysical assay identified compounds 20 and 21 as potent dual
HDAC/BRPF1 inhibitors with inhibitory activities against HDAC8
(IC50 ¼ 443 and 956 nM, respectively) and BRPF1 (Kd ¼ 67 and
4080 nM, respectively) [69]. Unfortunately, compounds 20 and 21
showed disappointing anticancer activity against two tested AML
cell lines, THP-1 and HL-60. This may be due to their poor cell
membrane permeability.
3. Dual inhibitors targeting histone erasers
3.1. Dual inhibitors targeting LSD1 and HDAC
3.1.1. Crosstalk between LSD1 and HDAC
LSD1 (lysine-specific demethylase 1), also known as KDM1A, is
the first discovered histone demethylase that requires FAD as a
cofactor to exert its demethylase activity. LSD1 specifically erases
methylation marks from mono- and dimethylated lysine 4 in his-
tone 3 (H3K4me1/2), leading to transcriptional silencing [70]. By
interacting with androgen receptor or estrogen receptors, LSD1 can
demethylate mono- and dimethylated lysine 9 in histone 3
(H3K9me1/2), thereby promoting gene transcription. LSD1 also
demethylate nonhistone substrates, such as p53 [71], DNMT1 (DNA
methyltransferase 1) [72], E2F1 (E2F transcription factor 1) [73],
and MYPT1 (myosin phosphatase targeting subunit 1) [74]. LSD1 is
significantly overexpressed or dysregulated in multiple types of
cancer, such as prostate cancer [75], small cell lung cancer [76],
colon cancer [77], breast cancer [78], and AML [79,80]. It has been
implicated in many cellular processes such as EMT (epithelial-
mesenchymal transition) [81], cell proliferation and differentiation,
control of stemness [82], autophagy [83], and malignant trans-
formation. Also, high LSD1 levels frequently correlate with poor
clinical outcomes in various cancers. RNAi-mediated knockdown or
10
European Journal of Medicinal Chemistry 222 (2021) 113588
pharmacological inhibition of LSD1 demonstrated anti-
proliferation and anti-metastasis effects in diverse cancers.
Studies from Shi's group indicate that genetic or pharmacological
abrogation of LSD1 can elicit potent antitumor immunity and T-cell
infiltration in low or non-immunogenic tumors. This is done
through an induced expression of endogenous retroviruses and
reduced expression of the RNA-induced silencing complex [84].
Therefore, developing small-molecule inhibitors of LSD1 has been
an attractive area of anticancer drug discovery [78], and numerous
LSD1 inhibitors have been developed over the last decade.
Till date, seven LSD1 inhibitors, including IMG-7289,
GSK2879552 [76], TAK-418 [6], ORY-2001 [85], INCB059872
[86,87], CC-90011 [88], and SP-2577 (Seclidemsta) [89], are being
tested in I/II phase trial for AML and solid tumors (Fig. 11) [90,91].
IMG-7289, GSK2879552, TAK-418, ORY-2001, and INCB059872 are
irreversible LSD1 inhibitors derived from tranylcypromine (TCP or
PCPA), a mechanism-based covalent monoamine oxidase MAO in-
hibitor with moderate activity against LSD1. Mechanistically, TCP-
based LSD1 inhibitors inactivated LSD1 through covalent modifi-
cation of FAD in the amine oxidase domain active pocket. CC-90011
and SP-2577 are highly potent reversible LSD1 inhibitors developed
by Celgene Corporation and Salarius pharmaceuticals, respectively.
Recent structural and functional studies have uncovered the
coordinated crosstalk between LSD1 and HDACs. First, LSD1 and
HDAC1/2 often function as a catalytic component in the same
transcriptional repressor complexes, including NuRD (nucleosome
remodeling and deacetylase), CoREST (corepressor of RE1 silencing
transcription factor) [92], and Sin3A (SIN3 transcription regulator
family member A), and exert their demethylase and deacetylase
activities in a mutually dependent manner. Using real-time NMR
assay, Song et al. confirmed that LSD1 tethered HDAC1 by RCOR1
scaffold protein to form a stable ternary CoREST complex and the
two enzymes were functionally related. However, they are located
at opposite ends of the complex. Inhibiting or activating one
enzyme significantly influenced the activity of the other enzyme
[93]. LSD1 regulated embryonic stem and carcinoma cells' plurip-
otency through HDAC1 mediated deacetylation of H4K16 interde-
pendently through CoREST complex. LSD1 coordinates with the
SIN3A/HDAC complex to repress a series of proapoptotic genes and
maintain sensitivity to chemotherapy in breast cancer [94].
Second, the direct interaction of HDACs with LSD1 in the cell
nucleus has been confirmed. HDAC1 deacetylated LSD1 at K374 in
the substrate-binding domain, subsequently facilitated the LSD1/
H3 effective binding and promoted its demethylation activity,
leading to a more permanent silencing of target genes (Fig. 12A)
[95]. Huang et al. demonstrated that LSD1 and HDAC5 were coor-
dinated overexpression in human breast cancer cell lines and
clinical patient samples. HDAC5 physically interacted with LSD1
and promoted LSD1 stability and demethylation activity through
up-regulating USP28, a bona fide deubiquitinase of LSD1, thereby
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al. European Journal of Medicinal Chemistry 222 (2021) 113588

Fig. 10. Reported HDAC/BRPF1 dual inhibitors.

Fig. 11. LSD1 inhibitors undergoing clinical trials.

11
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
facilitating breast cancer progression (Fig. 12B). Conversely, siRNA
depletion of HDAC5 significantly downregulated LSD1 protein
stability and suppressed breast cancer cell proliferation and inva-
sion [96]. Inactivating HDAC5 with sulforaphane, a natural bioac-
tive HDAC inhibitor, also led to a profound, decreased expression of
LSD1 protein through HDAC5 modulated LSD1 ubiquitination sys-
tem [97]. Additionally, the acetylation level for residues proximal to
H3K4 can differently influence LSD1 activity. The treatment of
cancer cells with HDAC inhibitors suppressed the expression of
LSD1 and promoted the H3K4 methylation and H3 acetylation.
Third, HDAC inhibitors were capable of synergizing with LSD1
inhibitors to suppress various malignancies [98e100]. In MDA-MB-
231 breast cancer cells, Huang and Co. found that shRNA-mediated
LSD1 depletion conferred enhanced antineoplastic effect of sulfo-
raphane. Cotreatment with sulforaphane and LSD1 inhibitor HCI-
2509 synergistically suppressed breast cancer cell line prolifera-
tion, including MCF10A-CA1a, MDA-MB-231, and MDA-MB-468,
except for normal breast epithelial cells. Moreover, in a thymic
nude mice engrafted with MDA-MB-231 cells, combined therapy
with sulforaphane and LSD1 inhibitor was significantly superior to
treatment with sulforaphane or HCI-2509 alone in inhibiting tumor
growth [97]. Another research work from the same group showed
that combined treatment with LSD1 inhibitor, Pargyline, and HDAC
inhibitor, SAHA, also led to superior growth inhibition and
apoptotic death in triple-negative breast cancer cells [101]. Pan-
HDAC inhibitor, Panobinostat, interacted synergistically with
Fig. 12. Molecular crosstalk model between HDAC1/5 and LSD1. (A) HDAC1 deacetylates LSD1 at K374, which results in proper LSD1/H3 binding and demethylation of H3K4 to
repress target genes. HDAC inhibitors stimulate LSD1 acetylation, leading to decreased LSD1 activity, up-regulated H3K4 methylation, and derepression of target genes; (B) HDAC5
facilitates stability and demethylase activity of LSD1 through up-regulating the expression of LSD1 deubiquitinase USP28, while HDAC5 inhibitors promote LSD1 degradation
through down-regulating USP28 expression in an HDAC5-dependent manner.
12
European Journal of Medicinal Chemistry 222 (2021) 113588
LSD1 inhibitor, SP-2509, in inhibiting AML cell growth (both
cultured and primary AML blasts). Simultaneous HDAC and LSD1
inhibition with Panobinostat and SP-2509 showed greater efficacy
in prolonging the overall survival of mice bearing human AML xe-
nografts compared with each drug alone, without any sign of
toxicity [99]. A combination of GSK690 and JNJ-26481585 or SAHA
caused a synergistic induction of cell death in RMS cells by
changing the balance between pro- and antiapoptotic BCL-2 pro-
teins, thereby inducing the intrinsic mitochondrial apoptotic
pathway [102]. Chandra's group conducted a preclinical evaluation
of combined HDAC and LSD1 inhibition in glioblastoma (GMB)
[103,104]. Results revealed that the simultaneous treatment of GBM
cells with TCP and SAHA led to synergistic apoptotic cell death,
while there were no significant effects on normal cells [104]. Also,
combining TCP and SAHA showed efficacy in decreasing the
viability of patient-derived glioma stem cells. Particularly, the
combination treatment could reduce tumor burden and extend
overall survival in an orthotopic xenograft GBM mouse model. In
contrast, individual treatment with SAHA or TCP provided no
improvement in overall survival [103]. Welch et al. identified a
synergistic interaction of SP2509 with Romidepsin to inhibit the
growth of ewing sarcoma cell lines [100].
3.1.2. Reported LSD1/HDAC dual inhibitors
Considering the intimate crosstalk between HDAC and LSD1 and
the distinct benefits of combining HDAC inhibitors with LSD1
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
inhibitors, developing inhibitors that simultaneously target HDAC
and LSD1 represents an interesting anticancer approach. To date,
several dual LSD1/HDAC inhibitors have been reported by our and
other groups, and most dual LSD1/HDAC inhibitors were designed
by tethering known hydroxamic acid or benzamide group of HDAC
inhibitors to the covalent LSD1 inhibitor, TCP.
Corin (compound 22) is a potent LSD1/HDAC dual inhibitor
obtained by fusing the pharmacophore of an HDAC inhibitor
(Entinostat) and a TCP-based LSD1 inhibitor (Fig. 13) [105]. Corin
simultaneously blocked the LSD1 and HDAC1 activity of the CoREST
complex with an IC50 of 0.33 and 0.21 mM, respectively. Interest-
ingly, Corin displayed more robust and sustained inhibition of
HDAC1 than Entinostat alone or a combination of Entinostat and
Bizine in the CoREST complex, but not in the MiDAC complex
lacking an LSD1 subunit. Though, Entinostat was a reversible HDAC
inhibitor, irreversible inhibition of HDAC1 by Corin was observed.
Corin exhibited more powerful antiproliferative activities against a
panel of ten melanoma cell lines than MS-275 at 1.0 mM, especially
in WM983B MM cells (EC50 ¼ 95 nM), but showed no apparent
effects on primary human melanocytes. Corin also possessed
excellent antiproliferative potency against cutaneous squamous
cell carcinoma lines IC1 (IC50 ¼ 41 nM) and MET1 (IC50 ¼ 6 nM),
which was better than the mono-functional inhibitors or a com-
bination of MS-275 and LSD1 inhibitor, GSK2879552. Interestingly,
depleting CoREST1 or LSD1 conferred enhanced anticancer effi-
ciency to MS-275 in WM983B and HCT-116 cells, but did not alter
Corin's antiproliferative effects. This suggests that Corin may also
exert its anticancer potency by blocking HDAC in other contents.
Corin displayed good metabolic stability in human plasma, human
liver microsomes, and mouse liver microsomes. In SK-MEL-5 mouse
xenografts, treatment with Corin by ip daily administration at a
dose of 30 mg/kg for 28 d, caused a 61% reduction in tumor volume
without noticeable effect on body weight and blood cell counts,
while MS-275 was too toxic to experiment with the same dose.
Western blotting and qRT-PCR analysis of the tumor cells collected
at the end of the animal studies showed that the expression of Ac-
H3K9, H3K4me2, p21, CHOP, and MXD1 elevated in the Corin-
treated group. Also, immunohistochemistry staining revealed that
the expression levels of Ki-67 were downregulated in the tumor
tissues of the Corin treatment group.
Furthermore, the significant antitumor efficacy of Corin was
also confirmed in HSJD-DIPG007 diffuse intrinsic pontine gliomas
(DIPG) xenografts by Shi and Co [106]. Co-targeting LSD1 and
HDACs with Corin synergistically changed global histone methyl-
ation and acetylation and potently suppressed DIPG growth both
in vitro and in vivo by re-programming chromatin to inhibit cell
cycle, while promoting death and differentiation. Impressively, the
Fig. 13. Structure of LSD1/HDAC dual inhibitor, Corin.
13
European Journal of Medicinal Chemistry 222 (2021) 113588
expression of Corin-target genes was associated with prolonged
survival time in a DIPG patient cohort.
Our group reported various TCP derivatives as LSD1/HDAC dual
inhibitors. Compound 23 emerged as the most promising dual in-
hibitor, with potent inhibitory activity against HDAC1/2 and LSD1
with an IC50 value of 15 nM, 23 nM, and 1.20 mM, respectively
(Fig. 14). Compound 23 could efficiently inhibit tumor growth in
MGC-803, MCF-7, SW-620, and A-549 human cancer cell lines with
IC50 values ranging from 0.81 to 4.28 mM by decreasing the mito-
chondrial membrane potential and inducing cellular apoptosis. The
intracellular LSD1 and HDAC inhibitory activity of compound 23
was proven by validating the significant accumulation of H3K4Me2,
H3K9Me2, and Ac-H3 after treatment with compound 23 in MGC-
803 cells [107].
A recent study by Sadhu et al. reported novel LSD1/HDAC6 dual
inhibitors using TCP as an LSD1 inhibitor pharmacophore and
hydroxamic acid as an HDAC inhibitor pharmacophore (Fig. 14)
[108]. Out of approximately 50 new compounds tested, compound
24 was identified as the most active dual inhibitor. Compound 24
exhibited irreversible and potent LSD1-inhibitory activity
(IC50 ¼ 5 nM) and a high selectivity over the MAO-A
(IC50 ¼ 8600 nM) and MAO-B (IC50 ¼ 6600 nM). Compound 24
also displayed nanomolar activity against HDAC6 and HDAC8 with
an IC50 value of 48 and 71 nM, but had a low activity against other
HDAC isoforms (1, 2, 3, 5, 7, 9, and 10), exhibiting 70-fold selectivity
for HDAC6 against HDAC1 (IC50 ¼ 3400 nM). Pure enantiomers (1R,
2S and 1S, 2R) of compound 24 were also synthesized to assess the
difference in potency. Interestingly, they showed similar potency to
compound 24 in enzymatic potency and antiproliferative activity.
Consistent with the authors' predictions, compound 24 showed
remarkable antiproliferative activities against three multiple
myeloma cell lines, MM.1S (EC50 ¼ 2 nM), MM.1R (EC50 ¼ 6 nM),
and RPMI-8226 (EC50 ¼ 280 nM). Pharmacokinetic studies in mice
identified that compound 24 exerted good oral bioavailability of
44%. More significantly, compound 24 showed potent in vivo anti-
tumor efficacy in MM.1S xenograft models. Oral administration of
compound 24 at 25 mg/kg for 12 d showed a good TGI of 67.0%
without significant changes in body weight, which was superior to
single LSD1 inhibitor GSK-2879552 (25 mg/kg, PO, TGI ¼ 40%) and
HDAC6 inhibitor ACY-1215 (50 mg/kg, IP, TGI ¼ 36%).
By incorporating SAHA and TCP with various polyamine chains,
Milelli et al. synthesized various TCP-based LSD1/HDAC dual in-
hibitors (Fig. 14). Compound 25 showed good dual-inhibitory ac-
tivities with a Ki of 42.52 nM for HDAC1 and an IC50 value of
3.85 mM for LSD1. Compound 25 showed comparable cytotoxic
activity against MCF-7 (IC50 ¼ 39.6 mM) compared with SAHA
(IC50 ¼ 38.2 mM), while at 70.0 mM compound 25 displayed more
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
Fig. 14. Reported TCP-based LSD1/HDAC dual inhibitors.
pronounced anticancer effect than SAHA (68.6% vs 56.6% of dead
cells) [109].
4SC-202 (compound 26, also named domatinostat) is an orally
available non-TCP-based LSD1/HDAC dual inhibitor and is currently
undergoing phase I clinical trials (Fig. 15). 4SC-202 exhibited se-
lective inhibitory activities on class I-selective HDAC with an IC50
value of 1.20 mM, 1.12 mM, and 0.57 mM for HDAC1, HDAC2, and
HDAC3, respectively [110]. It also displayed inhibitory activity
against LSD1 with an IC50 value of 1.20 mM [111]. Numerous studies
have demonstrated that 4SC-202 exerted potent antitumor efficacy
against multiple cancer cell lines, including hepatocellular carci-
noma cell [112], medulloblastoma cell [111,113], colorectal cancer
cell [114], cutaneous T-cell lymphoma [115], oral squamous cell
carcinoma [22], and urothelial carcinoma cell [110]. Moreover, in
both syngeneic mouse models with CTL-high C38 or CTL-low CT26
tumors, treatment with 4SC-202 led to elevated expression of
cancer-germline antigens, antigen-presenting machinery genes,
and MHC class I and II molecules, along with cytotoxic T-cells (CTL)
infiltration, which reinforce immune responses against tumors.
4SC-202 significantly reduced tumor volume in both CT26 and C38
tumor models, whereas anti-PD-(L)1 antibody had no obvious tu-
mor inhibitory effect. When anti-PD-(L)1 antibody was added, 4SC-
202 showed enhanced anticancer effects compared with single-
agent therapy and substantially prolonged event-free survival
[116]. Notably, all event-free animals were completely tumor-free
at the end of the study. Currently, 4SC-202 and anti-PD-1 anti-
body Avelumab combination therapy, for patients with advanced
Merkel cell carcinoma progressing on anti-PD-(L) 1 is under phase
II clinical trial.
More recently, Our group designed and synthesized a series of
novel non-TCP based LSD1/HDAC bifunctional inhibitors by
replacing the amidoxime group of stilbene-based LSD1 inhibitor 27
reported by our group with a hydroxamic acid moiety [117]. The
representative compound 28 exhibited potent activity and high
selectivity against LSD1 over MAOA/B with IC50 of 1.64 mM. It also
14
European Journal of Medicinal Chemistry 222 (2021) 113588
possessed significant HDAC inhibitory activity against HDAC1,
HDAC2, and HDAC6 with IC50 values of 0.37, 0.40, and 0.015 mM,
respectively. Notably, it showed over 20-fold selectivity against
HDAC6 over HDAC1/2. Compound 28 displayed potent in vitro
anticancer potency against all tested seven gastric cancer cell lines
with IC50 values of 0.23e1.56 mM, more potent than ORY-1001 and
SP-2509. In addition, compound 28 significantly modulated the
expression of Bcl-2, Bax, Vimentin, ZO-1 and E-cadherin, induced
apoptosis, reduced colony formation and suppressed migration in
MGC-803 cancer cells. Preliminary ADME studies revealed that
compound 28 showed acceptable metabolic stability in human
liver microsomes with minimal inhibition of cytochrome P450s
[118].
3.2. Dual inhibitors targeting LSD1 and JmjC KDM
3.2.1. Crosstalk between LSD1 and JmjC KDM
The JmjC domain-containing histone lysine demethylases
represent the largest lysine demethylase superfamily, consisting of
20 members divided into five subfamilies (KDM2/7, KDM3, KDM4,
KDM5, and KDM6) [119]. Unlike LSD1, a FAD-dependent deme-
thylase, JmjC KDM, catalyze oxidative demethylation reactions with
Fe (II) and 2-oxoglutarate (2-OG) as cofactors. Mechanistically,
Fe(II)/2-OG complex reacts with molecular oxygen to yield carbon
dioxide, succinate, and reactive ferryl-oxo species, which hydrox-
ylates the methylated histone lysine residue to produce a hemi-
aminal intermediate. The unstable hemiaminal products then
spontaneously break into formaldehyde and the demethylated
lysine, while regenerating the active Fe(II) center. In contrast to the
restricted catalytic activity of LSD1 at only mono- and dimethylated
lysines, JmjC KDM can erase all three methyl marks on lysine res-
idues [120]. JmjC KDM are overexpressed in multiple malignancies,
stimulate cancer cell proliferation and invasion, and facilitate effi-
cient tumor growth, underscoring the potential of JmjC KDM as an
anticancer target [121,122]. Recently, significant progress has been
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
Fig. 15. Reported non-TCP-based LSD1/HDAC dual inhibitors.
made in developing JmjC KDM inhibitors; most of them are
mechanism-based 2-OG competitive inhibitors and Fe2þ chelators
[123]. However, although some representative JmjC KDM inhibitors
showed potent anticancer activity in vivo, only a few of them have
been approved in preclinical or clinical trials due to poor selectivity
and toxicity.
JmjC KDM demethylates di- and tri-methylated marks on lysine,
including H3K4 and H3K9, while LSD1 is active on mono- or di-
methylated H3K4 and H3K9. Given the prevalence of H3K4Me3
and H3K9Me3, there should exist a functional connection between
LSD1 and JmjC KDM to accomplish H3K4 and H3K9 demethylation,
and research findings have confirmed the cooperation between
LSD1 and JmjC KDM to exert their demethylases activity.
In normal prostate and human prostate cancers, KDM4C co-
localized and interacted with LSD1 and androgen receptor (AR) to
assemble into a multiple-specificity demethylase complex, which
removed the repressive methyl marks from mono-, di- and trime-
thylated H3K9 to facilitate AR-dependent gene transcription [124].
KDM4C inhibitors were inactive against prostate LNCaP and colon
HCT116 cancer cells, but when combined with LSD1 inhibitor NCL-
2, they synergistically inhibited cancer cell growth [125]. KDM4C
and LSD1 were co-overexpressed and exerted an overlapping
oncogenic activity in human melanoma. KDM4C and LSD1 coop-
eratively blocked oncogenic Ras- or Braf-evoked senescence by
promoting the expression of E2F target genes, thus facilitating the
formation of aggressive melanomas. Pharmacological inhibition of
LSD1 or KDM4C effectively suppressed tumor growth in patient-
derived xenograft model of Braf-V600-mutant melanomas by
restoring senescence [126]. KDM4A and LSD1 were also identified
as AR coregulators to mediate AR-dependent transcription via
histone lysine-demethylation activity in human bladder cancer
[127]. Recent studies from Ray et al. showed that KDM4B formed a
protein complex with AP-2 family transcription factor, TFAP2C and
LSD1, which was enriched at the promoters of crucial trophoblast
genes to regulate active gene expression in trophoblast stem cells
[128]. Shang's group demonstrated that in breast cancers, KDM5B
15
European Journal of Medicinal Chemistry 222 (2021) 113588
physically interacted with LSD1 within the NuRD complex and
cooperatively regulated H3K4 demethylation. In the term of
mechanisms, KDM5B/LSD1/NuRD complex bound to target gene
promoters via H3K4me3 marks, which was initially recognized by
KDM5B, and the methyl groups were then sequentially erased by
the joint demethylase activities of KDM5B and LSD1, leading to
gene silencing [129]. Zhang et al. found that the co-overexpression
of LSD1 and KDM6B was tightly related to a bad prognosis in pa-
tients with head and neck squamous cell carcinoma (HNSCC).
Depleting LSD1 with siRNA potentiates KDM6B inhibitor activity in
HNSCC and vice versa. Combinational treatment with TCP and
KDM6B inhibitor GSK-J1 synergistically impaired tumor growth
and progression in the 4NQO-induced HNSCC model and FaDu
xenograft model in nude mice by inhibiting cell proliferation and
inducing cell apoptosis. In the xenograft model, the group treated
with TCP plus GSK-J1 showed a higher progression-free survival
than the single-agent treatment group [130].
Altogether, given the recent findings of their functional rela-
tionship, co-inhibiting JmjC KDM and LSD1 as a novel potential
therapeutic strategy for cancers have drawn increased attention.
3.2.2. Reported LSD1/JmjC KDM dual inhibitors
Mai et al. have disclosed various dual LSD1/JmjC KDM inhibitors
(pan-KDM inhibitors as called by the authors), which were
designed based on the LSD1 inhibitor, TCP, and linked with the 2,2-
bipyridine (29) or 5-carboxy-8-hydroxyquinoline (30) scaffolds,
two known 2-OG competitive templates developed for JmjC KDM
inhibition (Fig. 16) [131]. In all target compounds synthesized,
compounds 32 and 33 showed the most potent LSD1 inhibitory
activity with IC50 values below 1.0 mM and displayed greater than
40-fold selectivity over MAO-B, but these compounds had unsat-
isfactory activities against JmjC KMD with an exception for KDM4C
(IC50 ¼ 2.7 mM for 32 and IC50 ¼ 1.2 mM for 33). Compound 31,
having an IC50 value of 2.2 mM against LSD1, exerted low or sub-
micromolar activity against all tested JmjC KDM, but showed little
or no effect against other tested 2-OG enzymes lacking KDM
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
activity, such as hypoxia-inducible factor hydroxylases FIH and
PHD2. Compounds 32 and 33 induced more remarkable apoptosis
than single 29, 30, or TCP, or the combination of 29 and TCP in both
LNCaP prostate and HCT-116 colon cancer cells, with a concomitant
increase in H3K4Me2 and H3K9Me2/3, indicating effective dual-
target engagement. Nevertheless, compounds 32 and 33 have lit-
tle or no apoptotic induction ability in mesenchymal progenitor
noncancerous cells, suggesting anticancer selectivity.
Research by Benedetti et al. showed that compound 33 regu-
lated ERa signaling in breast cancer by inhibiting LSD1 and KDM6A.
In MCF-7 and tamoxifen-insensitive-ERa-positive BT474 cancer
cells, compound 33 strongly downregulated ERa expression at
protein and mRNA levels, accompanied by a significant growth
arrest and apoptosis. Conversely, compound 33 failed to reduce the
growth of triple-negative MDA-MB-231 cells, underscoring the
vital role of ERa regulation for its anticancer effect. Also, compared
with SAHA, 33 displayed less cytotoxicity to normal HaCaT cells. Its
in vivo efficacy was confirmed in both chicken embryo and mouse
models and ex vivo in human breast cancer specimens. In the
chicken embryo model, 33 reduced tumor size and abolished the
migratory potential of MCF-7 cells compared to tamoxifen. In hu-
man breast cancer specimens, compound 33 impaired cell growth
and suppressed ERa expression, recapitulating the observed effects
in MCF-7 cells. Its low cytotoxicity to healthy cells from isolated
neighboring tissue relative to breast specimens was also observed.
Oral administration of 33 also displayed good in vivo anticancer
activity with a TGI of 60% in MCF-7 xenografted nude mice without
obvious toxicity [132].
4. Dual inhibitors targeting histone erasers and writers
4.1. Dual inhibitors targeting HDAC and G9a
4.1.1. Crosstalk between HDAC and G9a
G9a, also known as euchromatic histone methyltransferase 2
(EHMT2) or lysine methyltransferase 1C (KMT1C), is the second
identified histone methyltransferase [133]. Using S-adenosylme-
thionine (SAM) molecule as the donor of the methyl group, G9a
Fig. 16. Reported dual LSD1/JmjC KDM inhibitors.
16
European Journal of Medicinal Chemistry 222 (2021) 113588
specifically catalyzes H3K9 to form H3K9me1 and H3K9me2, which
acts as a preferred binding site for heterochromatin protein 1 (HP1)
that recruits transcriptional repressors to exert transcriptional
silencing [134]. Apart from methylating histones, G9a has also been
described to methylate several non-histone proteins [135,136]. For
example, G9a can dimethylate the lysine-373 residue of tumor
suppressor gene p53, leading to transcriptional inactivation of p53
[137]. Under hypoxia, G9a can also methylate reptin at lysine 67,
facilitating the recruitment of HDAC1 to hypoxia-responsive gene
promoters to suppress the transcriptional activity of HIF-1a [138].
G9a plays an essential role in many biological processes, including
embryonic development [139], DNA damage repair [140], cell
growth, and autophagy [141].
Studies have reported that the expression levels of G9a are often
upregulated in many human cancers [142], including colorectal
cancer [143], breast cancer [144], ovarian cancer [145], liver cancer
[146], gastric cancer [147], hematological malignancies, and over-
expressed G9a is associated with both the occurrence and survival
of various cancers. Moreover, high G9a expression levels positively
correlate with poor survival [144,148e151]. Knockdown of G9a or
pharmacological inhibition of its activity significantly inhibited cell
proliferation and tumorigenesis [143,152]. Collectively, the crucial
roles of G9a in cancers suggest that G9a is a potential therapeutic
target for cancers [153].
To date, several G9a inhibitors have been discovered and re-
ported [152,154]. The best-studied G9a inhibitors are derived from
the first identified small molecule G9 inhibitor BIX01294 [155],
which is featured a quinazoline-amine scaffold (Fig. 17). Extensive
SAR exploration of BIX01294 displayed that modifications on the C2
position of the quinazoline ring were well tolerated. Also, G9a in-
hibitors with complete distinct skeleton have been identified, such
as aminoindole A-366, 2,4-diaminopyrimidine EPZ035544, 2-
aminobenzimidazole BRD4770, and quinolone CM-272.
Interestingly, G9a is shown to functionally interact with HDAC to
coordinate histone methylation and acetylation, promoting tran-
scriptional silencing of target genes. G9a and HDAC share several
common substrates, including H3K9 and p53K373. Acetylation of
H3K9 (H3K9Ac) is related to gene activation, while H3K9 me2/3
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
Fig. 17. Structures of representative G9a inhibitors.
represses gene expression. H3K9 in the acetylated state cannot be
further methylated by G9a, thereby contributing to indirect inhi-
bition of G9a. HDAC1 and G9a have been found in the same protein
complexes, including CoREST, C-terminal binding protein (CtBP)
[156,157]. The positive regulatory domain I-binding factor 1 (PRDI-
BF1) binds to the IFNB1 promoter and silences transcription in vivo
by recruiting G9a and HDAC2, and the presence of HDAC2 is
necessary for G9a-mediated methylation [158]. The growth factor
independent 1 (Gfi1) recruits G9a and HDAC1 to form a stable
repressive complex, which binds to the promoter of p21Cip/WAF1,
leading to decreased expression of p21Cip/WAF1 in HL-60 cancer cells
[159]. Hu et al. reported that G9a and HDAC interacted with Snail2
to form a multiprotein complex to exert the repression of E-cad-
herin transcription in hepatocellular [160] and lung carcinoma cells
[161], thereby promoting their migratory and invasive capacity.
Inactivating G9a or HDAC with small inhibitors could significantly
prolong survival time in mice harboring Snail2-overexpressing
cancer cells [160]. In breast cancer, Wang et al. found that HDAC1
served as a co-repressor with G9a and Yin Yang 1 (YY1) to silence
ferroxidase hephaestin, contributing to enhanced cellular labile
iron and increased proliferation of breast cancer in vitro and in vivo
[144]. Research by Seo's group documented that G9a cooperated
with both HDAC1 and HDAC2 and YY1 to repress JAK2 expression
during ATRA-mediated leukemia cell differentiation [162]. Also,
HDAC1 is functionally involved in G9a-mediated UHRF1 tran-
scriptional repression in 12-O-tetradecanoylphorbol-13-acetate
(TPA)-treated leukemia cells [163].
4.1.2. Reported HDAC/G9a dual inhibitors
Given the intimate association between G9a and HDAC in
regulating tumor suppressor genes, simultaneously targeting G9a
and HDAC may improve cancer therapy. Inspired by these pieces of
evidence, various novel G9a/HDAC dual inhibitors were designed
and synthesized by integrating the hydroxamic acid group into the
C2 position of BIX01294 (Fig. 18) [164]. Among those synthesized
target compounds, 34 was the most active compound against G9a
with an IC50 value of 7.14 mM determined using in-cell Western
blotting. Compound 34 also showed significant HDAC inhibition
with IC50 values of 13.8 and 5.73 mM against HeLa nuclear extracts
and K562 nuclear extracts, respectively. Compound 34 effectively
inhibited the proliferation of MDA-MB-231, MCF-7, and A549 with
IC50 values ranging from 10.02 to 37.36 mM, which is less potent
than SAHA and BIX-01294. Notably, compound 34 also displayed
17
European Journal of Medicinal Chemistry 222 (2021) 113588
much lower cytotoxicity in HEK293 with an IC50 value of 19.95 mM,
which is about ten times higher than that of SAHA (IC50 ¼ 2.48 mM)
and BIX-01294 (IC50 ¼ 2.05 mM).
4.2. Dual inhibitors targeting HDAC and EZH2
4.2.1. Crosstalk between HDAC and EZH2
EZH2 (zeste homolog 2 enhancer) is an essential histone
methyltransferase and a catalytic subunit of PRC2 (polycomb
repressive complex 2) [165]. EZH2 and two PRC2 core components,
EED (embryonic ectoderm development) and SUZ12 (suppressor of
zeste 12 protein homolog), primarily catalyze the trimethylation of
histone H3 lysine 27 (H3K27 me3) to silence PRC2 target genes
involved in-cell proliferation, cell-cycle regulation, cell differenti-
ation, and self-renewal. Apart from its transcriptional repressor
activity on chromatin, EZH2 also methylates non-histone targets to
regulate downstream genes. For example, EZH2/PRC2 can meth-
ylate cardiac transcription factor GATA4 at Lys299, thereby
decreasing p300-mediated GATA4 acetylation and attenuating
GATA4 transcription [166]. Overexpression and gain-of-function
mutations of EZH2 are frequently documented in various solid
and hematologic malignancies [167,168]. Also, elevated EZH2
transcript and protein levels are highly associated with tumori-
genesis, progression, and poor prognosis, making it a promising
epigenetic target for cancer therapy [169,170].
Currently, numerous small-molecule EZH2 inhibitors have been
developed [171]. Tazemetostat (EPZ-6438) was approved in January
2020 by the FDA for metastatic or locally advanced epithelioid
sarcoma [170]. GSK2816126, CPI-1205, CPI-0209, SHR2554,
HH2853, DS-3201b, and PF-06821497 are undergoing phase I/II
clinical trials as monotherapy and combined treatment for several
types of cancer (Table 3). Interestingly, most of the best charac-
terized EZH2 inhibitors are pyridone-based compounds, typically
featured with a pyridone main pharmacophore as the “head” re-
gion, an amide linker as the “neck” region, different aromatics as
the “body” region, and variable “tail” structures linked to the bot-
tom of the body. The pyridone moiety provides essential in-
teractions with the EZH2 domain in a SAM-competitive manner.
The“tail” structure was located in the solvent-exposed region,
making it a suitable position for further derivatization (Fig. 20)
[172].
Accumulating pieces of evidence showed that EZH2 cooperated
with HDAC to reprogram histone methylation and acetylation,
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al. European Journal of Medicinal Chemistry 222 (2021) 113588

Fig. 18. HDAC/G9a dual inhibitor based on BIX02194.

Table 3
Small-molecule EZH2 inhibitors undergoing clinical trials.

Drugs Structure Phase Disease Trial Number Status

Tazemetostat (EPZ-6438) Approved Advanced or metastatic epithelioid sarcoma NCT04204941 Completed

GSK2816126 (GSK126) Phase I Elapsed/refractory DLBCL NCT02082977 Terminated

Transformed follicular lymphoma
Non-Hodgkin's lymphomas
Multiple myeloma

CPI-1205 Phase I B-cell lymphomas NCT02395601 Completed

Phase I Advanced Solid tumors NCT03525795 Completed
Phase Ib/II Metastatic castration resistant prostate cancer NCT03480646 Active, not recruiting

CPI-0209 Structure not in public domain Phase I/II Advanced solid tumors NCT04104776 Recruiting
PF-06821497 Phase I Relapsed/Refractory small cell lung cancer NCT03460977 Recruiting
Castration resistant prostate cancer
Follicular lymphoma

Valemetostat (DS-3201b) Phase I Acute myelogenous leukemia or Acute lymphocytic leukemia NCT03110354 Recruiting
Phase I/II Recurrent small cell lung cancer NCT03879798 Recruiting
Phase II Relapsed or refractory adult T-cell Leukemia/Lymphoma NCT04102150 Active, not recruiting
Phase I Lymphomas NCT02732275 Recruiting
Phase II Relapsed/Refractory peripheral T-cell lymphoma NCT04703192 Not yet recruiting
Phase IIb Metastatic prostate, urothelial and renal cell cancers NCT04388852 Recruiting
SHR2554 Structure not in public domain Phase I/II Metastatic castration resistant prostate cancer NCT03741712 Recruiting
Phase I/II Advanced solid tumors NCT04407741 Recruiting
B-cell lymphomas
Phase I Relapsed or refractory mature lymphoid neoplasms NCT03603951 Unknown
HH2853 Structure not in public domain Phase I Non-hodgkin's lymphomas NCT04390737 Recruiting
Advanced solid tumors

leading to more permanent silencing of target genes. EZH2 was
shown to recruit HDAC1/2 to PRC2 through the EED protein, and
interdependently mediated transcriptional repression [173]. HDAC
inhibitors significantly attenuated EZH2 expression in both
cultured and primary AML cells, leading to decreased levels of
H3K27Me3/2 and hyperacetylation of H4, with a loss of clonoge-
netic survival [174]. Fukumoto et al. found that in ARID1A-mutant
ovarian cancers, HDAC2 interacted with EZH2 to repress the
expression of EZH2/ARID1A target proteins, including PIK3IP1, an
inhibitor of PI3K-AKT signaling path. Also, pan-HDAC inhibitor,
SAHA, treatment conferred enhanced tumor growth suppression
and improved survival to mice bearing ARID1A-mutant ovarian
tumors compared with the ARID1A wild-type ovarian tumors
(Fig. 19) [168]. Moreover, many synergistic antitumor effects have
been observed for EZH2 inhibitors combined with HDAC inhibitors.
In EGFR-wild-type and EGFR-mutant NSCLC cells, cotreatment with
EZH2 inhibitor, 3-deazaneplanocin A, and HDAC inhibitor, SAHA,
synergistically reduced EZH2 and SUZ12 expression, strongly
blocked the EGFR signaling pathway, and demonstrated superior
in vivo antitumor efficacy than each inhibitor alone in EGFR-
tyrosine kinase-resistant H1975 xenograft model [175]. Pollock
and Co. demonstrated similar EZH2 depletion upon HDAC inhibi-
tion in epithelioid sarcoma [176]. Lue et al. documented co-
exposure to Romidepsin and diverse EZH2 inhibitors (GSK126,

18
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
Fig. 19. Molecular crosstalk model between HDAC and EZH2 in ARID1A-mutant ovarian cancers. In ARID1A-mutated ovarian cancer cells, HDAC2 interacts with EZH2 to suppress
the expression of EZH2/ARID1A target tumor suppressor genes such as PIK3IP1, leading to enhanced cell proliferation and reduced apoptosis. HDAC2 inhibition or knockdown
derepresses the expression of PIK3IP1, inhibits cell growth and induces apoptosis.
EPZ011989, and CPI-1205) in lymphoma cell lines with EZH2 dys-
regulation displayed potent synergistic anticancer activity. Mech-
anistically, Romidepsin-GSK126 cotreatment disrupted the
assembly of PRC2 complex by acetylating RbAp46/48, a subunit
responsible for PRC2 complex recruitment to nucleosomes, leading
to decreased trimethylation of H3K27. In SU-DHL-10 mouse xeno-
graft models, dual therapy with GSK126 and romidepsin signifi-
cantly delayed tumor growth and improved overall survival
compared with single-agent treatment [177]. Wang et al. reported
that a combined treatment of either pan-HDAC inhibitor or a class I
HDAC inhibitor with EZH2 inhibitors displayed a robust synergistic
effect in inducing histone H3K27 acetylation and target gene
expression, leading to rapid induction of apoptosis and stronger
growth suppression of small cell carcinoma of the ovary and hy-
percalcemic type cells. Concordant with the potent in vitro anti-
cancer effects, synergetic in vivo efficacy between HDAC and EZH2
inhibitors were observed in the BIN67-xenograft models. Simulta-
neous treatment of EPZ-6438 and Quisinostat completely inhibited
xenografted tumor growth and significantly prolonged the median
survival time. In contrast, single treatment with EPZ-6438 or Qui-
sinostat failed to or only mildly suppressed the tumor growth,
respectively [178]. Grinshtein and Co. found that a combination of
EZH2 inhibitors (UNC1999, GSK126, and EPZ6438) and a unique
brain-penetrant class I HDAC inhibitor identified by Kim's group
demonstrated strong synergism in two brain-tumor initiating cell
lines by enhancing apoptosis and increasing DNA damage [179]. In
TGBC2TKB gallbladder cancer cells, SAHA treatment down-
regulated the expression levels of HDAC1, HDAC2, and EZH2. This
led to the simultaneous activation of p16INK4a, E-cadherin, and p21
and significant cell number decline. When combined with EZH2
siRNA treatment, a synergistic antiproliferative effect was observed.
Interestingly, no such findings were obtained in human gallbladder
epithelial cells, suggesting that the effect of SAHA depends on
EZH2-mediated tumor suppressor loss.
4.2.2. Reported HDAC/EZH2 dual inhibitor
Inspired by the above findings, Romanelli et al. developed a
first-in-class dual HDAC/EZH2 inhibitor, 36, by fusing the structural
features of SAHA through the “tail” of compound 35, a prototype of
the pyridone-based EZH2 inhibitor reported by their group (Fig. 20)
19
European Journal of Medicinal Chemistry 222 (2021) 113588
[180,181]. Compound 36 showed EZH2 inhibitory activity compa-
rable to compound 35, with an IC50 value of 7.37 mM. Concerning
HDAC inhibition, compound 36 displayed nanomolar potency
against HDAC6 (IC50 ¼ 5 nM) and sub-micromolar potency against
HDAC1 (IC50 ¼ 0.43 mM), HDAC3 (IC50 ¼ 0.45 mM), and HDAC8
(IC50 ¼ 0.11 mM). In leukemia U937 and rhabdomyosarcoma RH4
cancer cell lines, compound 36 was also an effective EZH2/HDAC
dual inhibitor by modulating known EZH2- and HDAC-dependent
histone markers, including H3K37Me3, Ac-H3, and Ac-tubulin.
However, compound 36 did not show exciting antiproliferative
activity in several tested cancer cell lines, including U937, RH4,
THP1, SH-N-SK, and U87, with IC50 values from 9 to 25 mM. Treating
RH4 cancer cells with compound 36 induced apoptosis, caused cell
cycle arrest in the subG1 phase, and promoted the expression of cell
surface adhesive receptors, CD11b and myogenin, markers of cell
differentiation and myogenic differentiation, respectively. Also,
compound 36 upregulates E-cadherin expression without affecting
the snail level, thereby contributing to the suppression of EMT
process in U937 cells.
4.3. Dual inhibitors targeting LSD1 and G9a
In the process of pursuit of novel non-covalent LSD1 inhibitors
by Speranzini et al., several well-characterized G9a inhibitors
featured with quinazoline skeleton were found to exhibit LSD1
inhibitory activity. The representative compounds 37e40 with IC50
values of 0.18, 0.18, 1.2, and 41.3 mM, respectively, toward G9a, are
shown in Fig. 21 [182]. Compounds E11 and MC2694 showed the
highest binding affinity to LSD1 with thermal shifts of 6.5  C and
4.5  C, respectively. Their potent LSD1 inhibitory activity was
further confirmed by enzymatic and fluorescence polarization as-
says, showing Kd values of 243 nM and 385 nM, respectively. In
MV4-11 leukemia cells, compound E11 did not induce any signifi-
cant increase in H3K4me2. However, qPCR analysis revealed that
E11 could strongly up-regulate the expression levels of Gfi1-B, an
established target of LSD1, indicating the effective inhibition of
intracellular LSD1. This potent dual LSD1/G9a inhibition of E11
translated into promising antiproliferative effects on MV4-11 with
an IC50 value of 0.89 mM.
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
Fig. 20. A pyridone-based HDAC/EZH2 dual inhibitor.
Fig. 21. Structure of LSD1/G9a dual inhibitors.
5. Dual inhibitor targeting histone writer and reader
5.1. Crosstalk between BRD4 and CBP/p300
Cyclic adenosine monophosphate-response element (CREB)-
binding protein (CBP) and adenoviral E1A binding protein of
300 kDa (p300) are highly homologous lysine acetyltransferases.
CBP and p300 proteins contain a single bromodomain adjacent to
the histone acetyltransferase catalytic domain, thus functioning as
an acetyllysine “writer” and “reader.” As an acetyllysine “writer,”
CBP/p300 can acetylate lysine residues on histone tails to remodel
chromatin in a relaxed superstructure, and lysine residues on non-
histone proteins to modulate their activities. Alternatively, as an
acetyllysine “reader,” CBP/p300 binds to acetylated histones and
non-histone proteins via their bromodomain, leading to the
recruitment of cellular transcriptional machinery to regulate gene
expression like c-Myc. Recent studies have demonstrated that
deregulation of CBP/p300 was closely related to the development,
progression of various cancers, and tumor immunity. Given the
vital role of CBP/p300 in driving cancer progression, depleting CBP/
p300 levels exert potent therapeutic effects in cancers. CBP/p300 is
considered a potential anticancer target, and several CBP/p300 in-
hibitors targeting HAT domains or bromodomains have been re-
ported recently. CCS1477 is the most advanced compound in this
20
European Journal of Medicinal Chemistry 222 (2021) 113588
field, tested in phase Ib/IIa clinical trials for treating hematological
malignancies and advanced drug-resistant prostate cancer [183].
As important bromodomain-containing acetyllysine “readers,”
it is not surprising that an interplay exists between CBP/p300 and
BRD4. Recent studies have confirmed that BRD4 and CBP/p300
played an important role in regulating the well-known oncopro-
tein, c-Myc. Wu et al. found that in embryonic stem cells, BRD4
interacted with CBP/p300 through its two bromodomains to rein-
force the histone acetyltransferase activity of CBP/p300, leading to
augmented acetylation of H3K27 and H3K56. Moreover, BRD4-
P300 interaction recruits chromatin remodeler, Brg1, to modulate
the chromatin structure, contributing to the transcription of
pluripotency-associated genes, including Oct4 and Nanog in em-
bryonic stem cells (Fig. 22) [184]. In NMC driven by BRD4-NUT
fusion oncoprotein, BRD4-NUT recruited and activated p300 via
the NUT portion to facilitate histone hyperacetylation and subse-
quent BRD4-NUT recruitment to newly acetylated chromatin,
leading to uncontrolled spreading of BRD4-NUT across chromatin.
Although BRD4 inhibitors showed limited efficacy against NMC due
to a narrow therapeutic window, combined treatment with CBP/
p300 inhibitor, GNE-781, and BRD4 inhibitor, OTX015, coopera-
tively depleted c-Myc and synergistically inhibited NMC growth
[185].
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
Fig. 22. Molecular crosstalk model between BRD4 and P300. In embryonic stem cells, BRD4 partners with P300 and enhances its histone acetyltransferase activity. Augmenting
histone acetylation by BRD4-P300 interaction recruits the chromatin remodeler Brg1 to favor an open chromatin environment, thus faliticating the expression of pluripotency-
associated genes including Oct4 and Nanog.
5.2. Reported BRD4-CBP/p300 dual inhibitor
NEO2734 (compound 41) is a first-in-class and orally active dual
epigenetic inhibitor, exhibiting a high affinity to BRD4 and CBP/
p300 with Kd values ranging from 3.5 to 31 nM (Fig. 23) [186,187].
In vitro cell growth inhibition screening against a panel of 60 cell
lines derived from various cancer tissues was conducted by Ber-
toni's group. Results obtained demonstrated that NEO2734
exhibited promising antiproliferative activity in multiple cell lines,
especially in leukemia and lymphoma with median IC50 values of
280 nM and 300 nM, respectively, which are more potent than the
BRD4 inhibitor, iBET-762. In vivo, the antitumor efficacy of NEO2734
was evaluated in an ABC-DLBCL lymphoma xenograft cancer model
at a dose of 10 mg/kg administered orally. NEO2734 significantly
induced tumor regression without apparent body weight loss, and
displayed higher efficacy than the BRD4 inhibitor, OXT-015, given at
a higher dose of 50 mg/kg. Moreover, in an MV4-11 xenograft NOD/
SCID mouse model, oral administration of NEO2734 even at a dose
of 1 mg/kg for 14 d inhibited tumor growth. Treatment at a dose of
3 mg/kg or 10 mg/kg enhanced tumor-repressive effect and was
more potent than OXT015 at 10 mg/kg [187].
It has been reported that SPOP hotspot mutations, such as F133V
and W131R, disturbed its binding interactions with BRD4 proteins,
resulting in decreased ubiquitination-mediated degradation and
accumulation of BRD4 proteins, thus conferring high-level resis-
tance to BRD4 inhibitors in prostate cancer. NEO2734 is active in
hotspot mutant (F133V) and non-hotspot mutant (Q165P) PCa cells
in vitro and in vivo. NEO2734 induced marked suppression of Q165P
and F133V C4-2 xenograft growth at a dose of 30 mg/kg, five days a
week for three consecutive weeks. Also, NEO2734 showed strong
antitumor activity against 16 MM cell lines with a median IC50 value
of 140 nM. This is more potent than the single BRD4 inhibitors,
iBET-762 and iBET-151, and the CBP/P300 inhibitor, CPI-637 [188].
The anticancer efficacy of NEO2734 was also impressive in NMC
Fig. 23. The structure of BRD4-CBP/p300 dual inhibitor, NEO2734.
21
European Journal of Medicinal Chemistry 222 (2021) 113588
in vitro and in vivo. In vitro, NEO2734 potently inhibited BRD4-
NUTþ NMC cells at low nM IC50 values and induced squamous
differentiation. This observation is comparable with the clinical BET
inhibitor, i-BET-762; however, disseminated BRD4-NUTþ NMC
xenograft models treated with NEO2734 at a dose of 8 mg/kg
exhibited potent growth suppression and demonstrated markedly
prolonged survival with median survival more than 100 d for PER-
403 mice and 44 d for 10e15 mice. This is more potent than i-BET-
762 with median survival of 41.5 d and 36.5 d, respectively [185].
6. Discussion and conclusion
Epigenetic dysregulation plays a crucial role in cancer devel-
opment, progression, and acquisition of drug resistance. Epigenetic
modulators, especially histone epigenetic modulators, have
emerged as attractive therapeutic targets for cancer. After more
than 20 years of unremitting pursuit, remarkable advances have
been made in this field, with eight epigenetic inhibitors approved
for cancer treatment and numerous other candidates undergoing
clinical trials. Despite recent significant processes achieved in
epigenetic therapy, to date, approved epigenetic inhibitors have
mostly been confined to a subset of hematological cancers. Simul-
taneously, their anticancer effects on solid tumors are unsatisfac-
tory. Moreover, narrow safety margin, dose-limiting adverse
events, and drug resistance limit their potential clinical use. New
strategies are urgently needed to overcome these challenges.
Epigenetic modulators cooperatively or antagonistically regu-
late various histone modifications, creating a complicated network
for epigenetic transcription control during biological or pathogenic
development. Numerous combination therapies of two epigenetic
drugs with different action mechanisms are currently undergoing
exploration in preclinical or clinical trials to improve the effec-
tiveness of these single epigenetic target drugs in cancer therapy.
Another attractive strategy for epigenetic drug development is the
discovery of dual inhibitors targeting different epigenetic modu-
lators. Compared with combination therapies, dual-target drugs
have several potential advantages, such as more predictable phar-
macokinetics, better patient compliance, reduced administration
dosage and toxicities. Excitingly, several dual epigenetic target in-
hibitors have been validated, and some of them showed superior
in vivo antitumor efficacy against solid tumors compared to the
combination of two individual single epigenetic target drugs.
Notably, the example of NEO2734 emphasizes the dual epigenetic
target inhibition as a promising strategy to overcome drug
resistance.
Despite their evident progress, development of dual epigenetic
inhibitors remains a challenging task: the first critical challenge is
Y.-C. Duan, S.-J. Zhang, X.-J. Shi et al.
how to balance the potencies toward desired epigenetic modula-
tors at an appropriate level to maximize the synergistic therapeutic
efficacy in vivo and safty profiles. Ideally, the in vitro activities for
dual targets should be within an order of magnitude. However, in
most cases, it is not an easy task to achieve and potency against one
target needs to be compromised. Moreover, differences in target
occupancy, biodistribution of compounds and enzyme densities in
various tissues as well as time course of target inhibition pose
roadblocks to develop dual epigenetic inhibitors with balanced
in vivo activity [189].
Achieving satisfactory pharmacokinetic profiles without
compromising the activities of inhibitors will be another major
issue to solve. Most reported dual epigenetic inhibitors are
designed using pharmacophore-linked or -fused strategy, which
generally results in high molecular weight and unfavorable phar-
macokinetic properties. Actually, some reported dual epigenetic
inhibitors do not show superior therapeutic benefits to traditional
epigenetic drugs, this is most likely due to the poor pharmacoki-
netics of obtained molecules. Apart from careful SAR explorations,
pharmacophore-merged strategy and fragment-based screening
strategy which give compounds with small molecular size need to
be fully explored to get dual epigenetic inhibitors with favorable
pharmacokinetics [190].
Systematic studies need to be conducted to ascertain the
appropriate epigenetic target combinations that can achieve su-
perior therapeutic efficacy through synergies. So far, most
described dual epigenetic inhibitors has particularly capitalized on
the known HDAC inhibitors (especially alkyl hydroximic acid based
HDAC inhibitors) due to their proven anticancer efficacy, well-
studied SAR and high synthesis feasibility. However, the pharma-
cological liabilities of alkyl hydroximic acid scaffold often cause
short pharmacokinetic half-lives, poor oral bioavailability and
toxicity [191]. Although several dual epigenetic inhibitors that do
not involve HDAC, such as NEO2734, have been reported, these
molecules are mainly discovered by serendipity. It is significant to
explore effective strategies and techniques that can help efficiently
design dual epigenetic inhibitors directed to diverse target com-
binations. Epigenetic combination therapy results from clinical
observations or preclinical/preliminary explorations provide proof
of concepts for the rational design of dual epigenetic inhibitors. For
example, BET inhibitor synergistic therapies with DOT1L inhibitor
or EZH2 inhibitor have achieved preliminary success in preclinical
studies. Future attempts could use this information to pursue novel
dual epigenetic target inhibitors.
Considering the tight connections between histone epigenetic
modulators and the proven anticancer potency of reported dual
epigenetic inhibitors, the development of dual epigenetic target
drugs will become a research hotspot. We foresee that the
continuous deciphering of the epigenetic networks in cancer will
further spark the discovery of dual epigenetic target inhibitors
soon.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Project No. U1704184); Science and Tech-
nology Tackling Key Project of Henan Province (Project No.
182102310214); Natural Science Foundation of Henan Province
(Project No. 202300410325).
22
European Journal of Medicinal Chemistry 222 (2021) 113588
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